Neoplasia is a disease characterized by an abnormal proliferation of cell growth known as a neoplasm. Neoplasms may manifest in the form of a leukemia or a tumor, and may be benign or malignant. Malignant neoplasms, in particular, can result in a serious disease state, which may threaten life. Significant research efforts and resources have been directed toward the elucidation of antineoplastic measures, including chemotherapeutic agents, which are effective in treating patients suffering from neoplasia. Effective antineoplastic agents include those which inhibit or control the rapid proliferation of cells associated with neoplasms, those which effect regression or remission of neoplasms, and those which generally prolong the survival of patients suffering from neoplasia. Successful treatment of malignant neoplasia, or cancer, requires elimination of all malignant cells, whether they are found at the primary site, or have extended to local/regional areas, or have metastasized to other regions of the body. The major therapies for treating neoplasia are surgery and radiotherapy (for local and local/regional neoplasms) and chemotherapy (for systemic sites) (Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991).
Despite the various methods for detecting, diagnosing, and treating cancers, the disease remains prevalent in all segments of society, and is often fatal. Clearly, alternative strategies for detection (including the development of markers that can identify neoplasias at an early stage) and treatment are needed to improve survival in cancer patients. In particular, a better understanding of tumor suppressors, and tumor-suppression pathways, would provide a basis from which novel detection, diagnostic, and treatment regimens may be developed.
The p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress (Levine, A. J., p53, the cellular gatekeeper for growth and division. Cell, 88:323-31, 1997; Giaccia and Kastan, The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12:2973-83, 1998; Prives and Hall, The p53 pathway. J. Pathol., 187:112-26, 1999; Oren, M., Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 36031-034, 1999; Vogelstein et al., supra). p53 is the most commonly mutated gene in human cancers, with more than 50% of tumors displaying some alteration in p53 (Hollstein et al., New approaches to understanding p53 gene tumor mutation spectra. Mutat. Res., 431:199-209, 1999; Hollstein et al., Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res., 22:3551-55, 1994).
Wild-type p53 has been called the guardian of the genome, as it responds to DNA damage or checkpoint failure by either arresting the cell in the G1 phase for damage repair or initiating an apoptotic pathway to eliminate the damaged cell entirely (Lane, D. P., Nature, 358:15-16, 1992; Levine, A. J., supra). p53 is also critical for maintenance of genomic stability, aberrant ploidy, gene amplification, increased recombination, and centrosomal dysregulation—all of which have been observed in cells lacking functional p53 (Donehower et al., Nature, 356:215-21, 1992). These data suggest that abrogation of p53 function is critical in tumorigenesis of cancer. Additionally, numerous studies indicate that inactivation of the p53 pathway is a pivotal event in tumorigenesis of all kinds of human cancers, including breast cancer (Vogelstein et al., Surfing the p53 network. Nature, 408:307-10, 2000). Accumulating evidence further indicates that, in cells that retain wild-type p53, other defects in the p53 pathway play an important role in tumorigenesis (Prives and Hall, supra; Oren, M., supra).
p53 is a short-lived protein whose activity is maintained at low levels in normal cells. The molecular function of p53 that is required for tumor suppression involves the ability of p53 to act as a transcriptional factor in regulating endogenous gene expression. A number of genes which are critically involved in either cell growth arrest or apoptosis have been identified as p53 direct targets, including p21CIP1/WAF1, Mdm2, GADD45, Cyclin G, 14-3-3σ, Noxa, p53AIP1, and PUMA (Nakano and Vousden, PUMA, a novel proapoptotic gene, is induced by p53. Molecular Cell, 7:683-94, 2001; Yu et al., PUMA induces the rapid apoptosis of colorectal cancer cells. Molecular Cell, 7:673-82, 2001; Oda et al., Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science, 288:1053-58, 2000a; Oda et al., p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell, 102:849-62, 2000b; el-Deiry et al., WAF1, a potential mediator of p53 tumor suppression. Cell, 75:817-825, 1993; Wu et al., The p53-mdm-2 autoregulatory feedback loop. Genes Dev., 7:1126-32, 1993; Barak et al., mdm2 expression is induced by wild type p53 activity. EMBO J., 12:461-68, 1993; Kastan et al., A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 71:587-97, 1992; Okamoto and Beach, Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J., 13:48 16-22, 1994). Furthermore, tight regulation of p53 itself is essential for its effect on tumorigenesis and the maintenance of normal cell growth.
Numerous studies imply the existence of multiple pathways involved in p53 stabilization (Shieh et al., DNA damage-induced phosphorylation of p53 alleviates inhibition MDM2. Cell, 91:325-34, 1997; Appella and Anderson, Signaling to p53: breaking the posttranslational modification code. Pathol. Biol. (Paris), 48:227-45, 2000; Ashcroft et al., Regulation of p53 function and stability by phosphorylation. Mol. Cell Biol., 19:1751-58, 1999; Blattner et al., DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation. Oncogene, 18:1723-32, 1999; Dumaz and Meek, Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J., 18:7002-10, 1999; Ashcroft et al., Stress signals utilize multiple pathways to stabilize p53. Mol. Cell Biol., 20:3224-33, 2000). However, the precise mechanism by which p53 is activated by cellular stress is not completely understood; it is generally thought to involve mainly post-translational modifications of p53, including phosphorylation, acetylation, and ubiquitination (Appella and Anderson, Signaling to p53: breaking the posttranslational modification code. Pathol. Biol. (Paris), 48:227-45, 2000; Giaccia and Kastan, The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12:2973-83, 1998). For example, in response to DNA damage, p53 is phosphorylated at multiple sites; these phosphorylation events promote p53 stabilization by preventing binding with Mdm2, thereby rendering p53 more resistant to Mdm2-mediated degradation (Shieh et al., supra; Appella and Anderson, supra).
By serving as a signal for specific cellular-protein degradation, ubiquitination plays a critical role in physiological regulation of many cellular processes (Hershko et al., The ubiquitin system. Nat. Med., 6:1073-81, 2000; Laney and Hochstrasser, Substrate targeting in the ubiquitin system. Cell, 97:427-30, 1999; Kornitzer and Ciechanover, Modes of regulation of ubiquitin-mediated protein degradation. J. Cell. Phys., 182:1-11, 2000). The ubiquitination of p53 was first discovered in papilloma-virus-infected cells, through the functions mediated by the viral E6 protein (Scheffner et al., The HPV-16 E6 and E6-AP complex functions as an ubiquitin-protein ligase in the ubiquitination of p53. Cell, 75:495-505, 1993). However, in normal cells, Mdm2 functions as a ubiquitin ligase (E3) that directly mediates p53 ubiquitination and subsequent degradation (Haupt et al., Mdm2 promotes the rapid degradation of p53. Nature, 387:296-99, 1997; Kubbutat et al., Regulation of p53 stability by Mdm2. Nature, 387:299-303, 1997; Honda et al., Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett., 420:25-27, 1997). Furthermore, by inhibiting Mdm2-mediated ubiquitin ligase activity, the p14ARF tumor suppressor can stabilize p53 in vivo in response to oncogene activation (Sherr and Webber, The ARF/p53 pathway. Curr. Opin. Genet. Dev., 10:94-99, 2000).
As indicated above, evidence suggests that, in cells that retain wild-type p53, other defects in the p53 pathway may play an important role in tumorigenesis. To date, at least one method of treating cancer has been developed that targets the p53 pathway. This treatment involves the stabilization of p53 by inhibiting Mdm2-mediated deubiquitination. It is estimated that 15-30% of all tumor cases exhibit overexpression of Mdm2. However, this enzyme is notoriously difficult to inhibit. Moreover, recent studies imply the existence of an alternative mechanism for p53 stabilization that may function even when the Mdm2-mediated ubiquitination pathway is intact (Ashcroft et al., 1999, supra; Blattner et al., supra; Dumaz and Meek, supra; Ashcroft et al., 2000, supra). Accordingly, while regulation of the p53 pathway is of intense interest, and presents a potential means of diagnosing and treating cancers, a greater understanding of this pathway and the regulation of p53 ubiquitination would provide a valuable basis upon which new diagnostic and therapeutic methods may be developed.